| Objective:To explore the role of lipoxygenase 15B(15-LOX-2)gene and 15-LOX-2 metabolite 15(S)-HETE in osteoclasts and osteoblasts related to the development of osteoporosis mechanism.Method:1.Effect of 15-LOX-2 on osteoclast function and its mechanism(1)Construction of 15-LOX-2 knock-down stable cell line from mouse precursor osteoclasts15-LOX-2 knockdown stable cell lines were constructed on mouse Raw264.7 cells and divided into three groups:shRen(control),shl5-LOX-2.1252 and sh15-LOX-2.2865(n=3).(2)Effect of 15-LOX-2 knock-down on osteoclast function in miceOsteoclast models were examined using anti-tartrate acid phosphatase(TRAP)staining.Osteoprotegerin(OPG),nuclear factor of activated T cells 1(NFATC1),Cathepsin K(CTSK)and C-Junexpression in osteoclasts were measured by WB.(3)Effects and mechanisms of 15-LOX-2 metabolite 15(S)-HETE on mouse osteoclastsThe osteoclast model was established with wild-type Raw264.7 and treated with absolute ethanol(solvent),estradiol(E2),arachidonic acid(AA)and 15(S)-HETE.Then ER-a,C-Jun and CTSK expression were detected by WB;E2+15(S)-HETE and E2+AA groups were increased on the basis of the above administration,and apoptosis was detected by flow cytometry;mRNA levels of osteoclast-associated genes Cxcl10,Mef2a,Pparg,and Runx2 were detected by RT-qPCR.2.Effect of 15-LOX-2 on the function of osteoblasts and its mechanism(1)Effects and mechanisms of 15-LOX-2 metabolite 15(S)-HETE on mouse osteoblasts3T3-E1 cells were induced into osteoblasts.The experimental grouping was the same as that in 1.(3).The cell models were examined by alkaline phosphatase(ALP)staining and alizarin red staining.The expression levels of osteoblast-related proteins,including epidermal growth factor receptor(EGFR),estrogen receptor 1(ER-a),C-Jun and CTSK were detected by WB.Besides,the mRNA levels of osteoblast-related genes Alpl,Kdm4b,Bglap,Col1a1 and Mef2a were detected by RT-qPCR.(2)Effects and mechanisms of 15-LOX-2 metabolite 15(S)-HETE on human osteoblastsPrimary Human Mesenchymal Stem Cells(HMSC)were induced into osteoblasts and grouped as before.The cell model was examined by ALP staining.In addition,the expression levels of EGFR,C-Jun and CTSK were examined by WB.Result:1.Effect of 15-LOX-2 on osteoclast function and its mechanism(1)Successful construction of mouse precursor osteoclast 15-LOX-2 knock-down stable cell lineThe results of flow cytometry after drug screening showed that the proportion of GFP in shRen,sh15-LOX-2.1252 and sh15-LOX-2.2865 cells was all about 95%.(2)The 15-LOX-2gene knockdown promotes the differentiation of osteoclasts in miceOsteoclast-induced cells were positive for TRAP staining;WB detection showed that compared with the shRen group,the expression of OPG in the sh15-LOX-2.1252 and 2865 groups decreased to 22%and 31%,respectively,both NFATC1 and CTSK were significantly increased(n=3,P<0.05).(3)The 15-LOX-2 metabolite 15(S)-HETE inhibits the differentiation of mouse osteoclastsAfter the osteoclasts were given solvent,E2,AA and 15(S)-HETE,the results of WB showed that the expression of ER-ain the E2 and 15(S)-HETE groups was lower than that in the solvent group(P<0.05),while there was no difference in the expression of ER-α in the AA group;CTSK and C-Jun protein expressions were slightly increased in the three groups,with 15(S)-HETE being the most pronounced.In the detection of the interaction between 15(S)-HETE and E2,the flow cytometry results showed that the early apoptosis ratio of the E2 group was 1.15 times that of the control group,15(S)-HETE was 1.28 times,and E2+15(S)-HETE was 2.39 times(P<0.05).RT-qPCR results showed that the expression of genes that promote osteoclast differentiation and activity,including Cxcl10 and Mef2a,in the E2 group and 15(S)-HETE group were significantly lower than those in the solvent group(P<0.05).E2+15(S)-HETE group made the expression of Mef2a lower than that of the two drugs alone,and also significantly decreased the expression of Runx2(P<0.05).Meanwhile,the AA group and E2+AA group significantly decreased the expression of osteoclast-related genes(P<0.05).2.Study on the effect of 15-LOX-2 on the function of osteoblasts and its mechanism(1)The 15-LOX-2 metabolite 15(S)-HETE promotes osteoblast differentiation and maturation in miceOsteogenesis-induced 3T3-E1 was positive by both ALP and alizarin red staining.After the model cells were treated with the corresponding reagents,WB showed that the expression levels of EGFR,ER-a,and C-Jun in the E2 group were increased.However,compared with the 15(S)-HETE and AA groups,there was no significant difference.RT-qPCR results showed that for genes promoting osteoblast differentiation,Alpl expression was significantly increased in the E2 group and 15(S)-HETE group compared with the control group(P<0.05).E2+15(S)-HETE group significantly increased the expression of AlplandKdm4b(P<0.05).The similar effects were observed in the AA group and E2+AA group.While for genes that inhibit osteoblast maturation,compared with the solvent group,the E2 and 15(S)-HETE groups significantly decreased Bglap,Col1a1,and Mef2a expression(P<0.05),furthermore,E2+15(S)-HETE similarly decreased their expression(P<0.05).In addition,the effect of AA was consistent with the E2 group(P<0.05).Whereas the E2+AA group only caused Bglap,Collal significantly decreased(P<0.05).(2)The 15-LOX-2 metabolite 15(S)-HETE promotes differentiation of human osteoblastsAfter induction of HMSC osteogenesis,the ALP staining results were positive.After administration of the aforementioned treatment in induced human osteoblasts,CTSK were slightly downregulated and C-Jun was slightly elevated in both E2 and 15(S)-HETE group,and no significant changes in EGFR were seen.Besides,several protein changes were not significant in the AA cell group.Conclusion:(1)The 15-LOX-2 gene and its metabolite 15(S)-HETE may slow the process of osteoporosis by regulating osteoclast-associated protein expression and inhibiting bone resorption.(2)15-LOX-2 regulates osteoblast differentiation and promotes the bone formation process through its metabolite 15(S)-HETE.(3)The 15-LOX-2 metabolite 15(S)-HETE synergizes the biological effects of estrogen(E2)on osteoblasts and osteoclasts,and enhances the anti-osteoporotic effect of E2. |
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